The invention relates to the field of immunology and vaccine development, in particular to the development of vaccines based on native antigen oligomers.
Many surface exposed proteins of pathogens and tumour cells are functional as oligomers. Examples for pathogens are for example, influenza virus hemagglutinin (HA) which occurs in virions as a homotrimer, and neuraminidase (NA) as a homotetramer. Also, the human immunodeficiency virus (HIV) glycoproteins gp140/gp120 is a homotrimer in its active form. Likewise, the respiratory syncytial virus (RSV) glycoproteins G and F occur in virions as tetrameric and trimeric homo-oligomers, respectively. Likewise, coronavirus spikes consist of homotrimers as is the case for the human respiratory coronaviruses and for SARS coronaviruses. Examples for tumour cells are for example, receptor tyrosine kinases (RTKs). RTKs are the high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. Growth factor receptors include: epidermal growth factor receptor, fibroblast growth factor receptors and platelet-derived growth factor receptor. Hormone receptors include: androgen receptor and estrogen receptor. An example of the epidermal growth factor receptor (EGFR) is the ErbB protein family of four structurally related RTKs. The four members of the ErbB protein family are capable of forming homodimers, heterodimers, and possibly higher-order oligomers upon activation by a subset of potential growth factor ligands. ErbB-1 is overexpressed in many cancers. The platelet-derived growth factor receptor (PDGF) family consists of PDGF-A, -B, -C and -D, which form either homo- or heterodimers (PDGF-AA, -AB, -BB, -CC, -DD). The four PDGFs are inactive in their monomeric forms. Such oligomeric proteins of pathogens and tumour cells are often involved in the pathogenicity and immunogenicity of the disease-causing entity (viruses, bacteria, parasites) or in cancer development, respectively, and are therefore important targets for vaccine development.
However, during vaccine preparation the integrity and, hence, the antigenicity of these oligomeric structures may be negatively affected e.g. if the manufacturing process involves inactivation of the pathogen. Alternatively, the oligomeric status of the antigen may not be obtained due to the production process used for the manufacturing of (recombinant) subunit vaccines. In both cases, this may result in aggregation or dissociation into a monomeric and/or misfolded state of the proteins.
Conformational epitopes embedded in the quaternary structures of oligomers may critically contribute to immunogenicity (Weldon et al. PLoS One 5 [2010], pii: e12466). Du et al. (Virology 395 [2009], 33-44) e.g. found that immunization of rabbits provided no evidence that trimerized gp140 constructs induced significantly improved neutralizing antibodies to several HIV-1 pseudoviruses, compared to gp140 lacking a trimerization motif. On the other hand, Grundner et al. (Virology 331 [2005], 33-46) showed that immunization of rabbits with the gp140 trimer elicited neutralizing antibodies of greater potency and breadth than did either gp120 or solid-phase proteoliposomes containing a cleavage-defective Env. Also Wei et al. (J. Virol. 82 [2008], 6200-6208) demonstrated that trimeric viral spikes serve as the optimal protein immunogens to elicit neutralizing antibodies against H5N1 isolates. Of other note, Bosch et al. (J. Virol. 84 [2010], 10366-10374) provided evidence that the combination of soluble trimeric HA and tetrameric NA of pandemic swine-origin 2009 A(H1N1) influenza virus in the presence of adjuvant provides protection against infection in ferrets.
Therefore, it is conceivable that the presence of native oligomeric protein antigens in vaccines is pivotal for their protective capacity. In many instances, oligomerization is determined by a subdomain having strong oligomerization properties. Also in many cases, such oligomerization subdomain of vaccine antigens is embedded in a membrane. To enable oligomerization of vaccine subunit antigens in the absence of a lipid environment, native oligomerization subdomains can be substituted by a heterologous coiled-coil motif with similar conformation-inducing properties. Examples of such motifs that have been successfully applied to obtain native oligomeric protein structures are a 32-amino-acid form of the GCN4 transcription factor (GCN), a 27-amino-acid trimerization domain from the C-terminus of bacteriophage T4 fibritin (T4F), and a soluble trimerization domain of chicken cartilage matrix (CART) protein (Selvarajah et al., AIDS Res. Hum. Retrovir. 24 [2008] 301-314; Yang et al., J. Virol. 74 [2000], 5716-5725; Yang et al., J. Virol. 76 [2002], 4634-4642).
Hence, technology to produce native oligomeric subunit vaccine antigens is available. Nevertheless, it is known that soluble subunit vaccine antigens are poorly immunogenic in general and need adjuvants and/or a particulate carrier system in order to raise robust immune responses. In the recent past there has been a growing interest in the development of novel non-replicating antigen presentation systems in order to increase the immunogenicity of antigens that could be used as vaccines. Many of these systems are designed in such a way to present the antigen as a polyvalent particulate structure. Some of the well appreciated examples are those of hepatitis B virus core and surface proteins genetically fused to foot-and-mouth disease virus (FMDV) (Clarke et al., Nature 330 [1987], 381-384) and HIV (Michel et al., Proc. Natl. Acad. Sci. USA 85 [1988], 7957-7961; Schlienger et al., J. Virol. 66 [1992], 2570-2576) antigens; the development of Ty virus like particles (VLPs) as antigen carriers (Adams et al., Nature 329 [1987], 68-70) where antigens are genetically fused to the C-terminus of the TYA gene encoded protein of the yeast retro-transposon Ty to form hybrid Ty-VLPs, parvovirus like particles (Miyamura et al., Proc. Natl. Acad. Sci. USA 91 [1994], 8507-8511). These technologies ensure that the antigen in question is presented in multiple copies in relatively large particles.
Other known particulate carriers for antigens are virosomes which are complexes composed of lipids and at least one viral envelope protein, produced by an in vitro procedure. The lipids are either purified from eggs or plants or produced synthetically, and a fraction of the lipids originates from the virus providing the envelope protein. Essentially, virosomes represent reconstituted, empty virus envelopes devoid of the nucleocapsid including the genetic material of the source virus(es). Virosomes are not able to replicate but are pure fusion-active vesicles. Known virosomes for use as antigen carrier include virosomes termed immunopotentiating reconstituted influenza virosomes (IRIVs). IRIVs are spherical, unilamellar vesicles with a mean diameter of 150 nm and comprise a double lipid membrane, consisting essentially of phospholipids, preferably phosphatidylcholines (PC) and phosphatidylethanolamines (PE). IRIVs may contain the functional viral envelope glycoproteins HA and NA intercalated in the phospholipid bilayer membrane. The biologically active HA does not only confer structural stability and homogeneity to virosomal formulations but also significantly contributes to the immunological properties by maintaining the fusion activity of a virus.
Although these known technologies can provide particulate carriers which enhance the immunological properties of a vaccine preparation, the methodologies are typically rather cumbersome and require specialized equipment and personnel. In addition, the use of viral material as carrier is preferably to be avoided. Furthermore, none of the existing technologies have been reported to be applied successfully in the manufacture of native oligomeric subunit vaccines.
Whereas it is known in the art (see e.g. WO 02/101026) to present an antigen to the immune system by fusion to a peptidoglycan binding sequence and attachment to particles derived from a Gram-positive bacterium, this particulate carrier technology has thus far only been described and applied in relation to the presentation of monomeric antigens.
WO 99/25836 and Bosma et al. (Appl. Environ. Microbiol. 72 [2006], 880-889) teach that one LysM domain suffices to mediate antigen obtain binding to Gram-positive microorganisms and/or peptidoglycan microparticles (BLPs, formerly called GEMs). This approach was for example followed by Raha et al. (Appl. Microbiol. Biotechnol 68 [2005], 75-81) and Moeini et al. (Appl. Microbiol. Biotechnol 90 [2011], 77-88). However, antigen binding via only a single LysM domain is rather limited (Bosma et al.) and not very stable, as shown by Raha et al. (FIG. 6) who determined that approximately 30 to 45% of the initially bound antigen is lost after a storage period of 5 days. In agreement with that observation, Moeini et al. shows in FIG. 6 that approximately 40% of the antigen bound through a single LysM domain is lost after a storage period of 5 days.
The successful manufacturing of vaccines requires long term storage for several months or sometimes even for years. The use of a single LysM binding domain results not only in low binding yields (Bosma et al.) but also in low stability of the bound antigens (Raha et al., Moeini et al.). Thus, this approach is unsuitable for an economically viable vaccine production process for BLP-based vaccines, which require optimal loading of antigens to the particles, i.e. high loading yields, in combination with antigens that remain stably bound over a prolonged period.
Prior to the present invention, the commonly held view to improve the efficacy and stability of antigen binding was to increase the number of LysM domains. In fact, it was demonstrated in the art that consecutive LysM domains in a single construct (2 to 3 domains in line; in cis/intramolecular) provides the most optimal and stable binding. See FIG. 3 of Bosma et al., showing a steep increase in binding affinity by the addition of a second LysM domain, and the level was even higher than that of wild-type AcmA comprising 3 LysM domains in cis. However, the present inventors observed that the approach of using two (or more) consecutive LysM domains in a single construct does not yield the expected results in case of oligomeric antigen binding. This is because the LysM tandem repeat mediates such a strong binding that not only functional oligomers but also non-functional monomers are bound to the carrier. The presence of non-functional monomers in vaccines is highly undesirable because such a heterogeneous complex makes it more difficult and cumbersome to characterize formulated vaccines. Furthermore, non-functional monomers containing LysM tandem repeats compete strongly with the functional oligomers for the available binding sites on the BLPs. Most importantly, non-functional monomers in vaccines may impose a health risk for the vaccinated subject since it is known in the art that non-functional, improperly folded antigens can induce a detrimental immune response.
The inventors therefore aimed at providing stable native oligomeric subunit vaccines not only having improved immunogenicity but which can also be produced in a relatively easy and economically attractive manner. In particular, it was an object to increase the safety and efficacy of (current) vaccines in a simple and reliable manner while avoiding the use of pathogenic or otherwise unsafe starting materials.